Pharmaceutical preparations and medicine capable of generating and/or containing thrombin

ABSTRACT

The invention relates to a pharmaceutically active substance for producing a drug that is capable of generating thrombin or that contains thrombin and compositions comprising thereof. The pharmaceutically active substance contains (A) prothrombin (coagulation factor II) obtained from plasma or by genetic engineering, (B) coagulation factors V, VIII, IX, X obtained from plasma or by genetic engineering that at least partially may be present in their activated state, and coagulation factor XIa obtained from plasma or by genetic engineering, and (C) prion-safe, coagulation-promoting phospholipids, where the phospholipids are optionally contained in liposomes.

CROSS REFERENCE TO RELATED APPLICATION(S)

The present application is a continuation of International PatentApplication No. PCT/AT03/000204, filed Jul. 21, 2003, published inGerman on Feb. 5, 2004 as International Patent Publication No.WO04/011023, which claims priority to Austrian Application No. A1113/2002, filed Jul. 23, 2002, all of which are incorporated herein intheir entireties.

INTRODUCTION

The invention concerns a pharmaceutical preparation capable ofgenerating, and containing, thrombin, and medicines manufacturedthereof.

BACKGROUND OF THE INVENTION

When blood vessels are injured, blood escapes from the vascular spaceand coagulates. The injured blood vessels are closed by the coagulatingblood and, in that manner, protect the organism from massive blood loss.Blood coagulation is caused by the enzyme thrombin, which is generatedfrom its zymogen, prothrombin, and causes the transformation of theprotein fibrinogen, which is present in blood plasma, into insolublefibrin. The exiting blood coagulates within minutes, with only one sixthof the fibrinogen contained in plasma being transformed into fibrin.Thrombin generation continues in the coagulated blood, which stillcontains large amounts of fibrinogen, until all of the residualfibrinogen in the coagulated blood has been transformed into fibrin.This process of continued thrombin formation and coagulation proceedsslowly over a prolonged period of time and may take several hours untilit is completed.

When fibrinogen is transformed into fibrin, thrombin causesfibrinopeptides A and B to split or partially split from the two ends ofthe fibrinogen molecule, whereby fibrin monomers are formed, whichremain soluble for some time. These aggregate laterally to fibrils,forming a fibrin network in the further course (Blombäck). This fibrinnetwork, which has formed in the coagulated blood, adheres to theinjured tissue or wound bed and, in that manner, leads to hemostasis andwound closure.

Thrombin also causes the transformation of coagulation factor XIII, thezymogen of coagulation factor XIIIa, into a transglutaminase. The lattercross-links proteins such as fibrin, fibrin monomers, fibrinogen, andother proteins occurring in blood plasma by covalent bonds. Thecross-linking of fibrin, in particular, is of great importance for thestability of the nascent fibrin network (Siebenlist et al.).

Thrombin also transforms the thrombin-activatable fibrinolysis inhibitor(TAFI) into a carboxypeptidase (TAFIa), which splits carboxyterminallysine residues from fibrin and thereby inhibits the formation of thetissue-plasminogen-activator-plasminogen-fibrin-complex, which isnecessary for the transformation of plasminogen into plasmin (Booth).

In vitro, the formation of thrombin in blood or blood plasma and thusthe onset of the coagulation process is rendered possible by tissueextracts. Such extracts can best be obtained from brain using aqueousmedia or organic solvents (Morawitz).

The coagulation-active material, which can be extracted using a suitablebuffer and which is termed thromboplastin, consists of an apoprotein,the tissue factor, and coagulation-active lipids. Small amounts ofthromboplastin, when added to blood or plasma, suffice to generate rapidcoagulation.

Using organic solvents, it is possible to separate thecoagulation-active lipids from apoprotein. The aqueous apoprotein partis then termed partial thromboplastin, which, in the presence of kaolin,glass powder, and other surface-active substances also leads to rapidcoagulation when blood or blood plasma are added.

This has led to the concept of an activation cascade of enzymesresulting in the formation of the enzyme thrombin, which causes bloodcoagulation (Davie et al.; MacFarlane).

The coagulation process which is triggered by thromboplastin is referredto as the extrinsic pathway of blood coagulation, in contrast to thecoagulation process which is triggered by partial thromboplastin, thelatter being termed the intrinsic pathway. The two processes have incommon that they transform coagulation factor X into Xa, albeit bydifferent routes. Accordingly, a differentiation is made between theextrinsic and intrinsic tenase pathways in the first part of thecoagulation process, which leads to activation of coagulation factor X.In another enzyme complex, prothrombinase, coagulation factor Xaconverts prothrombin into thrombin. This process is referred to as thecommon pathway. The enzyme system which generates coagulation factor Xawith the aid of thromboplastin is termed the extrinsic tenase complex,in contrast to the enzyme system in which partial thromboplastin plays arole, i.e. the intrinsic tenase complex.

The prevailing view has it that tissue factor, jointly with coagulationfactor VIIa and thrombocytes (i.e., platelets) trigger blood coagulationafter injuries (Rapaport et al.). Tissue factor occurs in almost alltissues in highly variable amounts along with coagulation-active lipids,and the two substances, when in contact with blood, form the extrinsictenase complex, since small amounts of activated coagulation factor VIIare always present in blood (Drake et al.). The extrinsic tenase complextransforms both, coagulation factors X and IX into Xa and IXa,respectively, and IXa transforms X into Xa. The activation of factor Xand the resulting formation of thrombin, however, come to a halt rapidlyby the tissue factor pathway inhibitor (TFPI). The temporarily formedextrinsic tenase complex as well as coagulation factor XIa independentlylead to the activation of the intrinsic tenase complex, which, as far asthe activation of coagulation factor X is concerned, is 50-fold moreactive than the extrinsic tenase complex. The intrinsic tenase complexconsists of activated coagulation factors IXa, VIIIa, andcoagulation-active phospholipids and is not inhibited by TFPI (von demBorne et al.). In the intrinsic pathway, the activity of the enzymecoagulation factor IXa, which transforms coagulation factor X in Xa, isincreased 100,000 to 1,000,000-fold. This increase is caused byco-factor VIIIa, which itself is not an enzyme, and certainphospholipids at an optimum calcium ion concentration.

Since the intrinsic tenase complex transforms coagulation factor X intoXa very rapidly, and the latter, jointly with cofactor Va andcoagulation-active phospholipids, activates prothrombin, great amountsof thrombin are formed in a very short time. (Mann et al.).

The individual coagulation factors and platelets—the essentialcomponents of blood responsible for the process of coagulation—arenormally present in abundance. Only if one of these components isreduced by 90% or more, an increased propensity to bleeding can benoticed. Bleedings become life-threatening only in deficiency stateswhere a coagulation factor and/or platelets drop to several percent oftheir normal values. The central importance of tissue factor intriggering the coagulation process and its dissemination in all organsis beyond doubt, however, severe disturbances of blood coagulation occurin tissues with low tissue factor content in cases where a factor of theintrinsic tenase complex or of the prothrombinase complex ispathologically reduced. A case in point are patients suffering fromhemophilia A or B. The blood of these patients still coagulates in mostinstances, however, because the intrinsic tenase complex is deficient orabsent, thrombin formation in the coagulated blood is insufficient andthe clot dissolves rapidly, so that no satisfactory hemostasis isachieved.

Thrombin, mostly of bovine origin, is used as a medicinal product fornon-parenteral administration to achieve hemostasis in cases ofsuperficial injuries. Its hemostyptic effect could be improveddecisively when administered jointly with medicinal products containingfibrinogen (Grey; Young et al.). Because of their species-specific use,fibrinogen and thrombin are obtained primarily from allogenic sourcematerial today. By combining the application of thrombin withfibrinogen-containing medicinal products, one attempts to mimick andimprove the physiological blood coagulation and accompanying hemostasis.This can be achieved also in patients with severe blood coagulationdisturbances (Matras et al.).

By combining the application of fibrinogen concentrates, whosefibrinogen content amounts to 10- to 20-fold the fibrinogen content ofblood, and great amounts of thrombin (100-1000 Upper mL), it is possibleto reduce the coagulation time in such a fibrinogen-thrombin mixture toa matter of seconds and obtain a 10 to 100-fold reduction compared tothe physiological bleeding time. This has made it possible topractically achieve instantaneous hemostasis when suchfibrinogen-thrombin mixtures are applied in an optimal manner, providedthat no larger blood vessels, particularly arterial vessels, wereinjured (Spängler).

The fibrinogen transformed into fibrin by thrombin adheres to the woundbed as does coagulated blood, the transglutaminases which were activatedby the action of thrombin obviously causing covalent bonds between theinjured tissue and the fibrin formed. This strong adherence of theformed fibrin to tissue can be used also to glue non-bleeding tissue,since the formed fibrin does not impair the healing of the glued tissuein most cases and is largely degraded in a matter of days or weeks(Matras et al.).

When fibrinogen-thrombin mixtures are used to achieve hemostasis, an asrapid as possible initiation of the coagulation process is desirable. Incontrast, a gradual onset of the coagulation process is preferable incases where parts of tissue are glued and also for sealing purposes.This makes it possible for the tissue parts to be adapted moreappropriately, and similar adaptations are necessary in sealing. Thusfar, a slowing of the coagulation process has been achieved by areduction of the thrombin concentration to approximately 1% of theamount of thrombin used to achieve hemostasis. However, the use of both,high and low concentrations of thrombin is accompanied by disadvantages.

Highly viscous fibrinogen solutions containing between 5 and 10%fibrinogen can be brought to coagulate with thrombin amounts rangingfrom 100 to 1000 units within seconds. This short coagulation time isnecessary to achieve hemostasis rapidly after the application of such amixture to a bleeding site and arrest the bleeding. The disadvantage ofsuch a procedure is that the fibrinogen and thrombin solutions arepoorly mixed, since the high viscosity of the mixture does not allowsatisfactory mixing in a short time. What results is a non-homogenouscoagulation with an attendant impairment in biomechanical quality.

On the other hand, if fibrinogen-thrombin mixtures are used not toachieve hemostasis but rather to glue parts of tissue, it is necessaryin most cases to slow down the coagulation of the fibrinogen-thrombinmixture in order to be able to optimize the adaptation of the parts tobe glued or sealed before coagulation occurs. At present, this isattempted by reducing the amount of thrombin to between one tenth to onehundredth of the amount of thrombin used to achieve hemostasis at thedisadvantage that not all of the fibrinogen present is converted intofibrin and not all of the factor XIII is converted to factor XIIIa. Thisprocedure is also not suitable for obtaining an optimal fibrin clot,since the low thrombin concentration is not sufficient to transform TAFIinto TAFIa.

A further problem is the use of bovine materials for the manufacture ofmedicinal products containing thrombin. Since a risk of transmission ofprions by any bovine organs cannot be excluded with certainty, bovinethrombin is hardly used anymore today.

Bovine thrombin has the further disadvantage that it is antigenic forother species and can provoke allergies and anaphylaxes. In addition,patients treated with bovine thrombins have been observed to developcoagulation disorders, which is attributable to the fact that bovinethrombin can cause the formation of antibodies against coagulationfactors which cross-react with human coagulation factors, thus retardingthe coagulation process.

In manufacturing thrombin from prothrombin, thromboplastin from animalsource material, mostly bovine brain, has often been used to activatethrombin, which increases the yield. Because of the risk of transmissionof prions, thromboplastin from bovine source is rarely used anymore inthe manufacture of thrombin, and a poor thrombin yield is put up withinstead.

SUMMARY OF THE INVENTION

The invention relates to a pharmaceutically active substance forproducing a drug that is capable of generating thrombin or that containsthrombin. The pharmaceutically active substance comprises (A)prothrombin (coagulation factor II) obtained from plasma or by geneticengineering, (B) coagulation factors V, VIII, IX, X obtained from plasmaor by genetic engineering that at least partially may be present intheir activated state, and coagulation factor XIa obtained from plasmaor by genetic engineering, and (C) prion-safe, coagulation-promotingphospholipids, where the phospholipids are optionally contained inliposomes.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention aims inter alia at making available a virally safepharmaceutical preparation which is capable of generating thrombin and

which, when formulated into a medicinal product capable of generatingthrombin and when mixed with a pharmaceutical preparation containingfibrinogen, gives a complete and homogenous mixture of the twocomponents, without too rapid a coagulation process preventing completemixing. The aim, however, is not only to render possible completemixing, but also the adaptation of tissue parts to be glued or sealedbefore coagulation begins. Thrombin generation must continue also afterthe onset of coagulation in order to convert all of the fibrinogen,factor XIII, and TAFI into fibrin, factor XIIIa, and TAFIa,respectively, so that the wound closure obtained by fibrin be durable,and

in which the prothrombin it contains is converted as completely aspossible into thrombin and the latter can be further purified and usedfor the manufacture of a medicinal product containing thrombin. Thismedicinal product can be used as such or jointly with other medicinalproducts containing fibrinogen.

The medicinal products manufactured from the pharmaceutical preparationscapable of generating thrombin should contain the allogenic material ofthe species to which they are intended to be applied only. Likewise,only species-specific proteins should be used in their manufacture. Allsource and auxiliary materials used should exclude a risk oftransmission of prions. The generation of thrombin from prothrombin invivo and in vitro should be as complete as possible, since it is onlythe prothrombin present in an already formed fibrinogen clot from whichthrombin can be generated.

The pharmaceutical preparation or substance according to the inventionis suitable for the manufacture of a medicinal product containing, orcapable of, generating thrombin and is characterized in that itcontains:

-   prothrombin obtained from plasma or by recombinant technology

(coagulation factor II),

-   coagulation factors V, VIII, IX, X obtained from plasma or by    recombinant technology, which may be present at least partly in    activated form, and plasmatic or recombinant coagulation factor XIa,    and-   prion-safe, coagulation-active phospholipids, which may be    incorporated in liposomes.

The pharmaceutical preparation according to the invention containspreferably coagulation factor VII obtained from plasma or by recombinanttechnology or a mixture of plasmatic or recombinant coagulation factorsVII and VIIa. In addition, it contains preferably a recombinant tissuefactor as such or in relipidated form.

The pharmaceutical preparation according to the invention may alsoconsist of two or more plasma fractions and the coagulation activephospholipids, the individual plasma fraction containing one or morecoagulation factors and/or activated coagulation factors of components(A) and (B). The whole plasma fraction mixture must, however, containall coagulation factors and/or activated coagulation factors containedin components (A) and (B), tissue factor preferably being added.

The coagulation factors, in particular, are manufactured exclusivelyfrom the blood plasma of a particular mammalian species or thecorresponding recombinant coagulation factors or activated coagulationfactors, including tissue factor.

In the pharmaceutical preparation according to the invention,prothrombin, coagulation factors V, VIII, IX, X, XIa, and coagulationfactors VII and VIIa, if present, as well as tissue factor arepreferably rendered virally safe by virus inactivation and/or viruspartitioning. In a subset of embodiments, phospholipids may be of plantorigin or plant and synthetic origin, preferably in a ratio ofphosphatidylcholines to phosphatidylserines of 3:1 or ofphosphatidyl-cholines to phosphatidylserines tophosphatidylethanolamines of 2:2:1

The pharmaceutical preparation according to the invention may be presentin deep-frozen or freeze-dried form.

The invention also concerns a medicinal product capable of generatingthrombin which can be manufactured from a pharmaceutical preparationaccording to the invention.

The medicinal product capable of generating thrombin according to theinvention may be present in deep-frozen or in freeze-dried form.

A preferred embodiment of the medicinal product capable of generatingthrombin according to the invention is characterized in that it containsthrombin in such amounts that it will contain no more than 1 U ofthrombin per mL after thawing or reconstitution.

The invention also concerns a medicinal product containing thrombin andwhich can be manufactured from a pharmaceutical preparation capable ofgenerating thrombin according to the invention. It may be provided indeep-frozen or freeze-dried state.

The invention also concerns a medicinal product mixture which coagulateswithin 30 to 300 seconds by mixing a medicinal product according to theinvention with a medicinal product containing fibrinogen prior toapplication.

The invention also concerns a medicinal product containing thrombinwhich, if mixed with the medicinal product mixture described above willcoagulate within 3 to 10 seconds.

A preferred method for the manufacture of a pharmaceutical preparationcontaining thrombin according to the invention is characterized in thata pharmaceutical preparation capable of generating thrombin according tothe invention is mixed with Ca-ions, which results in a productcontaining thrombin and which is subsequently purified bychromatography.

A preferred embodiment of the process according to the invention is thatthe pharmaceutical preparation is rendered virally safe by subjecting itto virus inactivation during or after thrombin generation and to virusremoval after thrombin generation is complete.

The invention also concerns a process for the manufacture of apro-cofactor-concentrate containing coagulation factors V and VIII andwhich is characterized in that plasmatic cryoprecipitate is dissolvedand fibrinogen is separated from the solution, whereby a solution isobtained which contains coagulation factors V and VIII and which can bemixed with calcium chloride.

Another process according to the invention concerns the manufacture of aprothrombin complex concentrate containing coagulation factor XI inaddition to coagulation factors II, VII, VIIa, IX, X, and XI, andcontaining per 1.0 μg prothrombin at least 0.030 μg coagulation factorXIa and which is characterized in that plasmatic cryoprecipitatesupernatant is adsorbed onto an anion exchanger and a prothrombincomplex containing coagulation factor XIa is eluted after washing theadsorbate and storage in the cold.

The invention is based on the finding that a virally safe intrinsictenase-prothrombinase-complex is responsible for a next-to-complete,timewise controllable formation of a-thrombin from virally safeprothrombin. The intrinsic tenase-prothrombinase-complex is generated byactivation of the respective coagulation factors, which either are orhave been rendered virally safe, in the presence of optimalCa-ion-concentrations, with prion-safe, coagulation-active phospholipidsand the virally safe activator substance coagulation factor XIa and/orthe virally safe activator substances coagulation factors VIIa andtissue factor.

Thus, the aims of the present invention are accomplished by apharmaceutical preparation, consisting of three components (ComponentsA, B, and C), for the manufacture of a virally safe medicinal productcapable of generating, or containing, thrombin, the pharmaceuticalpreparation being characterized in that.

Component A contains virally safe prothrombin obtained from plasma orgenetically engineered, as a pharmaceutical active substance,

Component B contains virally safe, plasma derived or geneticallyengineered coagulation factors V, VIII, IX, X, XIa as pharmaceuticalactive substances and, in case the generation of extrinsic tenasecomplex is desirable in addition to the generation of prothrombinase andintrinsic tenase complexes, also contains virally safe coagulationfactors VII and/or VIIa as well as tissue factor as pharmaceuticalactive substances, and

Component C contains fully synthetic, coagulation-active phospholipidsas pharmaceutical active substances having a transformation point of theparacrystalline into the liquid-crystalline form at beyond 15° C. Amixture of the pharmaceutical active substances, i.e. the coagulationfactors and/or activated coagulation factors contained in Components Aand B can also be obtained by mixing two or more plasma fractionscontaining all necessary pharmaceutical active substances and to whichtissue factor has been added, if necessary.

The invention also concerns a process for the manufacture of apro-cofactor-concentrate containing coagulation factors V and VIII andwhich is characterized in that plasmatic cryoprecipitate is dissolvedand fibrinogen is separated from the solution, whereby a solution isobtained which contains coagulation factors V and VIII and which can bemixed with calcium chloride.

Another process according to the invention concerns the manufacture of aprothrombin complex concentrate containing coagulation factor XI inaddition to coagulation factors II, VII, VIIa, IX, X, and XI, andcontaining per 1.0 μg prothrombin at least 0.030 μg coagulation factorXIa and which is characterized in that plasmatic cryoprecipitatesupernatant is adsorbed onto an anion exchanger and a prothrombincomplex containing coagulation factor XIa is eluted after washing theadsorbate and storage in the cold.

The invention is based on the finding that a virally safe intrinsictenase-prothrombinase-complex is responsible for a next-to-complete,timewise controllable formation of α-thrombin from virally safeprothrombin. The intrinsic tenase-prothrombinase-complex is generated byactivation of the respective coagulation factors, which either are orhave been rendered virally safe, in the presence of optimalCa-ion-concentrations, with prion-safe, coagulation-active phospholipidsand the virally safe activator substance coagulation factor XIa and/orthe virally safe activator substances coagulation factors VIIa andtissue factor.

Thus, the aims of the present invention are accomplished by apharmaceutical preparation, consisting of three components (ComponentsA, B, and C), for the manufacture of a virally safe medicinal productcapable of generating, or containing, thrombin, the pharmaceuticalpreparation being characterized in that.

Component A contains virally safe prothrombin obtained from plasma orgenetically engineered, as a pharmaceutical active substance,

Component B is manufactured by mixing stock solutions of virally safecoagulation factors V, VIII, IX, X, XIa as well as coagulation factorsVII, VIIa, and tissue factor. The concentrations of the individualcoagulation factors in their stock solutions lie between 10 and 100units per mL; the tissue factor stock solution contains 25 μg/mL.Instead of a stock solution of coagulation factor V, a stock solution ofcoagulation factor Va may be used. The content of an individualcoagulation factor in a stock solution is determined using thecorresponding deficient plasmas, and a standard curve with pooled normalplasma is established to determine each individual coagulation factor.

In order to determine what amounts of the individual coagulation factorsare necessary to still obtain an optimum thrombin generation, differentmixtures of Components A and B are prepared, containing one unit each ofcoagulation factors II, V, VIII, IX, X, XIa, and II, XIa, VII, VIIa, and2.5 μg tissue factor, respectively, per mL of sample. After addition ofthe liposome emulsion and subsequent recalcification, thrombingeneration is determined at 26° C. at different time intervals. In thesamples, prothrombin consumption and the generation of β- and y-thrombinare also determined. Prothrombin is transferred into thrombin in thepresence of 0.1% PEG and the amounts of coagulation factors and tissuefactor per mL are determined which are necessary to still achieve anoptimum thrombin generation.

Mixtures of Components A and B may also be obtained from two or moreplasma fractions to the extent they contain as pharmaceutical activesubstances all coagulation factors and activated coagulation factorsnecessary for the generation of the intrinsic tenase and prothrombinasecomplexes. The intrinsic tenase pathway is activated either bycoagulation factor XIa or via the extrinsic tenase pathway, particularlywhen TFPI is absent. It is also possible to activate the intrinsictenase pathway by coagulation factor XIa and via the extrinsic tenasepathway simultaneously. In the presence of coagulation factor XIa, or byits addition to, a mixture of coagulation factors which are necessaryfor the generation of the intrinsic tenase complex, it is also possibleto determine whether or not at least 90% of the prothrombin in themixture have been transferred into thrombin, and if that is not thecase, what amounts of factor VIIa and tissue factor are necessary toachieve a complete transfer of prothrombin into thrombin. A surplus ofcoagulation-active phospholipids is used to generate thrombin. Theactivation is carried out at 26° C. or 37° C. and at an optimum Ca-ionconcentration.

The coagulation factors used and/or the plasma fractions containing thecoagulation factors as well as the genetically engineered tissue factorare rendered virally safe by virus inactivation, e.g. thesolvent/detergent method, and subsequent virus removal bynanofiltration.

Component C is manufactured from mixtures of fully synthetic, andtherefore, prion-safe, choline- or serinephospholipids or fromcholine-serine-ethanolamin-phospholipids. Only phospholipids are usedwhich transfer from the paracrystalline gel state into theliquid-crystalline state between 15° and 40° C. From the phospholipidmixtures, emulsions can be produced which contain liposomes havingdiameters ranging from 20 nm to 1000 nm and which have the polar partsof the phospholipids in an outer membrane. Using temperatures between50° and 100° C., liposomes can be produced having diameters of 20 nm-200nm. Such emulsions can be sterilized using bacterial-tight filters,sterile filled, dried, and stored under nitrogen. The freeze-driedemulsions can be reconstituted with water.

The coagulation factors used and the non-relipidated tissue factor canbe rendered virally safe by virus inactivation, preferably thesolvent/detergent method and subsequent removal of thesolvent/detergent. Quantifiable traces of the solvent used,tri-n-butylphosphate, and of the detergent, Tween 80, remain in thethrombin-containing solution. Virus removal is achieved preferably bynanofiltration after prior clarifying filtration using a series offilters with narrowing pores, decreasing from 5000 nm to 35 nm.Preferably, after filtration through a 35 nm filter, also nanofilterswith pore sizes of 20 nm and 15 nm may be used.

Thrombin generation of this pharmaceutical preparation is highlytemperature dependent. Therefore, it is possible to inhibit theformation of thrombin from prothrombin in mixtures of Components A, B,and C, even at optimum Ca-ion concentration, if low temperatures aroundfreezing-point are used in processing. The pharmaceutical preparationaccording to the invention can be deep-frozen for storage or can befreeze-dried and stored between 0° and 25° C.

The pharmaceutical preparation capable of generating thrombin can beformulated at low temperatures into a medicinal product having athrombin generation capacity of preferably 100 to 1000 units of thrombinper mL, sterile filtered, portioned, and filled.

The pharmaceutical preparation capable of generating thrombin is alsoused for the manufacture of thrombin-containing medicinal products.Thrombin generation is preferably carried out at temperatures rangingbetween 20° and 40° C.

For the manufacture of thrombin from a pharmaceutical preparationcapable of generating thrombin containing one or more components thathave not been virus inactivated, virus inactivation can be carried outduring or after thrombin generation. The virus inactivating substancescan be removed in the course of the subsequent purification of thegenerated thrombin. It is possible to obtain a thrombin-containingpharmaceutical substance having a thrombin content of 1000 to 10000units per mL, which is subjected to virus removal by clarifyingfiltrations and subsequent nanofiltration. The virally safe substancecan then be formulated, sterile filtered, and processed into a medicinalproduct.

Medicinal products capable of generating, and/or containing, thrombincan be used in combination with fibrinogen-containing medicinal productsto achieve hemostasis, to glue sutured tissue, and seal body cavitiesand vessels against leakage of gasses and body fluids.

By mixing a medicinal product capable of generating thrombin with afibrinogen-containing medicinal product and addition, if any, of lowamounts of thrombin, the coagulation time of such a mixture can beadjusted to 30 to 300 seconds. In that manner, complete mixing of themedicinal products in such a mixture is possible, even if the viscosityis high. Also, after complete mixing of the medicinal products, enoughtime remains for the necessary adaptation of the tissue parts that needto be glued or sealed. Once coagulation sets in—similarly to thephysiological coagulation process—more thrombin is generated in thepartly coagulated fibrinogen, so that after some time—up to severalhours—practically all prothrombin has converted into thrombin and thelatter transforms all of the fibrinogen into fibrin. Such a continuedthrombin generation renders possible not only the homogenous generationof fibrin in the already coagulated fibrinogen, but also the uniformactivation of factor XIII and subsequent onset of the cross-linking ofthe generated fibrin, as well as the activation of TAFI and thesubsequent splitting-off of the end-terminal lysine residues of fibrin.

The application of fibrinogen-containing mixtures for hemostasisrequires a very short coagulation time. In order to achieve this, and atthe same time guarantee a homogenous formation of the coagulatedfibrinogen and the activation of zymogens such as factor XIII and/orTAFI, fibrinogen-containing medicinal products can be mixed completelywith medicinal products capable of generating thrombin, and suchmixtures can be applied to the bleeding site jointly with a thrombinsolution having a high thrombin content. Although the onsettingcoagulation does not immediately lead to a homogenous fibrin clot, thegeneration of thrombin in the fibrin clot continues as thethrombin-generating medicinal product contained in the clot continues totransform prothrombin into thrombin, resulting in a homogenouscoagulated fibrin clot with homogenously distributed factor XIIIa andhomogenously distributed TAFIa. This makes it possible to achieve anexcellent fibrin structure and high resistance against fibrinolyticinfluences even with rapidly coagulating mixtures of thrombin-containingand fibrinogen-containing medicinal products.

Another possibility to trigger spontaneous thrombin generation in amixture of coagulation factors capable of generating thrombin whichconsists of prothrombin complex and factor VIII is by addition offactors of the contact system. For that purpose, coagulation factors XIand XII need to be added, unless they are already contained in themixture.

The activation of coagulation factor XI in vivo as yet is not understoodin all detail. What is known is that blood cells and cells of theendothelium play an important role in this activation process. In blood,factor XI is bound to high molecular kininogen (Kd10-8) and prothrombin(Kd10-7) in non-covalent form. These complexes may accumulate onactivated platelets, this leading to activation of coagulation factor XIif thrombin and/or coagulation factor XIIa are present.

In vitro, coagulation factor XI can be activated by coagulation factorXIIa on negatively charged surfaces, coagulation factor XIIa beinggenerated from coagulation factor XII by kallikrein. The formation ofkallikrein from prekallikrein present in blood can occur by differentmechanisms, such as by prekallikrein activator or metal salts of ellagicacid.

According to the invention, thrombin can be generated spontaneously in amixture of coagulation factors capable of generating thrombin andconsisting of prothrombin complex and coagulation factor VIII, byactivation of the coagulation factor XI contained therein, allcomponents of the mixture having been virus inactivated. Preferably,this activation is performed in the presence of prion-safe,coagulation-active phospholipids with virally safe kallikrein. Insteadof kallikrein, also virally inactivated prekallikrein with virallyinactivated prekallikrein activator or prekallikrein activators can beused, such as metal salts of ellagic acid.

For the manufacture of thrombin-containing pharmaceutical substances,virus activation can also be performed during or after thrombingeneration.

The following examples describe the invention in greater detail.

EXAMPLES

Preparation of Component A.

100 mg of virally inactivated prothrombin are dissolved in 1 L of 0.1%sodium citrate solution at pH 7.5, and the prothrombin content per mL isdetermined using prothrombin-deficient plasma. After the addition ofecarin, the generated amounts of meizothrombin and thrombin aredetermined by splitting of chromogenic substrate S-2238. In addition,thrombin generation is determined using a prothrombinase preparation.The generated thrombin is determined with the aid of a fibrinogensolution as thrombin time. With this fibrinogen solution, a standardcurve was previously established using the international standard forα-thrombin. This prothrombin stock solution can be deep-frozen at −20°C. and stored for at least six months.

Preparation of Component B from Individual Coagulation Factors.

Virally safe, highly purified coagulation factor concentratesmanufactured from plasma or obtained by genetic engineering aredissolved in 0.1% sodium citrate pH 7.5 and mixed under stirring: 3.2 mgof factor V, 0.2 mg of factor VIII, 5.0 mg of factor IX, 11 mg of factorX, and 0.5 mg of factor XIa. After sampling, the solution is deep-frozenand stored at −20° C. or temperatures below that. An equivalent sampleis used to determine the amount of a 1M CaCl₂-solution which isnecessary to adjust the Ca-ion-activity to 5 mM-8 mM.

Preparation of a Mixture of Components A and B.

The frozen solutions of Component A, prepared according to Example 1,and Component B, prepared according to Example 2, are carefully thawed.Care must be taken for the temperature not to rise above 4° C. From thesolutions thus obtained, a mixture of equal parts of Components A and Bis prepared, the necessary samples are drawn, and the mixture of the twoComponents is again frozen and stored at −20° C.

Preparation of a Mixture of Plasma Fractions Containing the CoagulationFactors of Components A and B.

From 10 L of deep-frozen Source Plasma that has been carefully thawed ata temperature not exceeding 4° C., the cryoprecipitate is separated fromthe cryo-supernatant by centrifugation at 6000 g for 15 minutes.

a. Prothrombin Complex Concentrate Containing Coagulation Factor XIa:

The cryo-supernatant is mixed with 16 g of weak anion exchangerDEAE-Sephadex A-50, the pH is adjusted to between 7.8 and 8.8 using 0.1normal sodium hydroxide, and the mixture is stirred at a temperature ofbetween 3° and 6° C. for one hour. The mixture can be kept at thistemperature until the ion exchanger is separated by filtration and/orcentrifugation, which must be accomplished within no more than 24 hours.The liquid released from the ion exchanger is stored for preparation ofother plasma fractions, and the separated ion exchanger is used for themanufacture of prothrombin complex. The ion exchanger obtained in themanner described can be stored at a temperature of between 4° and 6° C.for up to 100 hours. For further processing, the separated ion exchangeris washed twice, each time with 1 L of 0.5% saline, and theprothrombin-complex-containing coagulation factor XIa is obtained byelution of the ion exchanger with 1000 mL of a 0.3 NaCL solution at pH7.5. After elution, the ion exchanger is washed with 500 ml of a 0.3 MNaCl solution at pH 7.5 once more, and the rinse is added to the eluate.Using ecarin, the amount of prothrombin present in the total eluate isdetermined, and the total eluate is adjusted by dilution to the desiredprothrombin content, which may lie between 2 and 5 prothrombin units permL. The total eluate, which is coagulation factor-XIa-containingprothrombin complex concentrate, may be frozen and stored at −20° C.Using samples drawn before freezing, the contents of coagulation factorsand activated coagulation factors are determined.

b. Preparation of a Pro-Cofactor Concentrate:

The cryoprecipitate sedimented from plasma by centrifugation isdissolved in 750 mL of 0.3% citrate buffer pH 7.0 and mixed with 110 gglycine. After stirring at a temperature of between 0° and 2° C. for onehour, the precipitated fibrinogen is separated by centrifugation at 3000g for 15 minutes, and the supernatant, which contains coagulationfactors V and VIII, is frozen. In a sample of the supernatant which hasbeen diluted with water at a ratio of 1:10, the content of factor VIIIis determined, as well as the amount of CaCl₂ which is necessary forrecalcification, in order to obtain an ion activity equivalent to thatof a 5 mM CaCl₂ solution. The frozen solution can be stored at −20° C.

c. Preparation of a Mixture of Coagulation-Factor-XIa-ContainingProthrombin Concentrate and Pro-Cofactor Concentrate:

A recalcified sample of pro-cofactor concentrate prepared according toExample 4b. which has been diluted with distilled water 1:10, is addedto prothrombin concentrate prepared according to Example 4a. in amountsof 0.1, 0.2, 0.4, and 0.8 mL per mL of prothrombin concentrate. To eachof these mixtures, 0.1 mL of a liposome emulsion prepared according toExample 5 is added. After incubation at 26° C. for eight hours, theamount of pro-cofactor concentrate is determined which is necessary totransfer 90% or more of the prothrombin present into thrombin.

For the preparation of the mixture from prothrombin complex concentratecontaining coagulation factor XIa, and pro-cofactor concentrate whichhave been obtained from 10 L of deep-frozen plasma and have been storedfrozen, these concentrates are thawed and mixed at the optimum ratiopreviously determined. The mixture is frozen and stored at −20° C.

Manufacture of Component C Without Emulgator.

Fully synthetic, and therefore, virally and prion-safe phospholipids areused. 200 mg 2Na-1,2-di-oleoyl-sn-glycero-3-phospho-L-serine, 400 mg1,2,di-oleoyl-sn-glycero-3-phosphocholine, and 400 mgdi-oleoyl-ethanolamine-phospholipid are dissolved in 10 mL chloroform,and the solution is evaporated in a 250-mL round-bottomed flask byheating and continuous rotation until most of the chloroform is removed.Residual chloroform is removed by a nitrogen stream, and 10 mL of 0.1%citrate buffer solution pH 7.3 are added. The phospholipid film on theinner wall of the round-bottomed flask is completely emulsified in thebuffer solution at 65° C. During emulsification, the flask is repeatedlyshaken in a Vortex device until the lipid film has completelydisappeared. The emulsion as such or diluted 1:10 with 0.1% citratebuffer pH 7.0, is filtered through a filter having a pore size of 0.45μm and is subsequently sterile filtered through a 0.22 μm filter. Thisliposome emulsion can be stored sterile in a refrigerator and is shakenin a Vortex device for 1 minute prior to use. The emulsion is tested forsterility and pyrogenicity according to the European Pharmacopoeia (Eur.Pharm. 4^(th) Edition 2002, pages 123-126, 2.6.1. Sterility. Pages131-132, 2.6.8. Pyrogens).

Preparation of Component C Using Sodium Cholate.

250 mg 2Na-1,2-di-oleoyl-sn-glycero-3-phospho-L-serine, 750 mg1,2,di-oleoyl-sn-glycero-3-phosphocholine and 500 mg sodium cholate aredissolved in 10 mL of a 1+1 mixture of chloroform and methyl alcohol,and the two solvents are evaporated under vacuum so that a thin, uniformfilm settles on the inner wall of a round-bottomed flask. The film iswashed off with 10 mL 0.1% citrate buffer pH 7.3, and the emulsionobtained in that manner is processed and sterile filtered as describedin Example 5.

Virally safe activator substances for the activation of the intrinsictenase pathway.

Coagulation factor XIa alone or in combination with coagulation factorXI, or coagulation factor VIIa, preferably with tissue factor, are usedas activator substances. Coagulation factor XIa can also be used jointlywith coagulation factor VIIa and tissue factor. For use aspharmaceutical active substances, the activated coagulation factors andtissue factor are virus inactivated by a solvent/detergent method, andafter removal of the solvent/detergent, are subjected to viruspartitioning by nanofiltration.

The following pharmaceutical active substances, dissolved in 0.3%citrate buffer pH 7.3, are used as stock solutions of the activatorsubstances:

-   30 μg factor XIa/mL;-   30 μg factor XIa and 50 ng factor XI/mL;-   30 μg factor XIa and 20 ng factor VIIa/mL;-   30 μg factor XIa and 20 ng factor VIIa and 10 ng tissue factor/mL;-   10 μg/mL tissue factor.

For the purpose of determining the best suitable activator substance, 10μL each of a dilution series of the individual activator substances areadded to 1 mL each of the coagulation factor mixtures prepared accordingto Examples 3 or 4 and are mixed with a 10 μL of a 1:100 dilution of aliposome emulsion prepared according to Example 5. Afterrecalcification, the mixtures are kept at 26° or 37° C. The amount offactor Xa generated is determined using chromogenic substrate S-2251 atintervals of 10, 30, 60, and 120 min. The activator substance whichgenerates the greatest amount of factor Xa from the coagulation factorconcentrates produced according to Examples 3 and 4 afterrecalcification, is used for the manufacture of a pharmaceuticalsubstance capable of generating thrombin.

Determination of the amounts of Component C necessary to transform atleast 90% of the prothrombin present in a mixture of Components A and Bat 26° C. within eight hours or 37° C. within two hours.

Coagulation factor mixtures according to Examples 3 or 4, to whichactivator substances in amounts determined according to Example 7 mayhave been added, are mixed with increasing amounts of liposomeemulsions. To 10 mL-samples of each coagulation factor mixture, 0.1,0.2, 0.4, or 0.8 mL of a 1:100 dilution of a liposome emulsion producedaccording to Example 5 are added after recalcification, the mixtures areincubated for eight hours at 26° or two hours at 37° C. The minimumamount of liposome emulsion is determined which is necessary totransform at least 90% of the prothrombin present into thrombin.

Preparation of a virally safe pharmaceutical substance capable ofgenerating thrombin.

A virally safe coagulation factor mixture produced according to Example3 (Components A and B) is thawed, if necessary mixed with an appropriateamount of a virally safe activator substance selected as described inExample 7, and the mixture is stirred at 0° to 2° C. for 15 minutes. Tothe mixture, the amount of liposome emulsion (Component C) determined inExample 5 is added. The mixture is adjusted to a concentration of 0.9%NaCl using solid sodium chloride, subjected to clarifying filtration,and sterile filtered through 0.22 μm and/or 0.1 μm filters. The wholemanipulation, including sterile filtration, is carried out at atemperature between 0° and 2° C. The pharmaceutical substance capable ofgenerating thrombin obtained in that manner is sampled, frozen, andstored at −20° C. The necessary tests for stability and pyrogenicity arecarried out according the European Pharmacopoeia. The capacity ofgenerating thrombin is determined in in-process controls and in thesterile filtrate after recalcification at 37° C. over two hours, alongwith residual amounts of prothrombin which can be activated by ecarin,in order to guarantee an at least 90% conversion of prothrombin intothrombin.

Preparation of a virally safe pharmaceutical preparation containingthrombin.

The frozen mixture produced from a factor-XIa-containing prothrombincomplex concentrate and a pro-cofactor concentrate according to Example4 is thawed, and, if desirable after Example 7, mixed with a selected,calculated amount of activator substance. By addition of 10 mg Tween 80and 0.3 mg tri-n-butylphosphate per g protein of the coagulation factormixture at 26° C. after recalcification and addition of the amount ofliposome emulsion determined according to Example 6, the major amount ofthe prothrombin is converted into thrombin within eight hours, and virusinactivation is carried out at the same time.

The thrombin-containing solution can be either frozen and stored or beused immediately or after thawing. By the addition of sodium citratesolution, the Ca-ion concentration is adjusted to 25 mM and the pH to6.5. Under continued stirring at room temperature, polyethylene glycolhaving a molecular weight of between 6000 and 8000 is added in an amountto achieve a 0.1% solution. A 20% CM-sepharose suspension that has beenwashed with 0.025 M sodium citrate pH 6.5 is then added under stirringuntil at least 95% of the available thrombin has been adsorbed onto theCM-sepharose. The required amount of CM-sepharose is determinedpreviously by estimating the lowest amount of the 20% emulsion whichadsorbs at least 95% of the available thrombin. The CM-sepharose iscentrifuged off between 3000 and 5000 g for 30 minutes, the sediment isresuspended in 25 mM citrate buffer pH 6.5 and filled into anappropriate column. The column is washed at pH 6.5 with 1% citratebuffer containing also 0.1% PEG and eluted with a sodium chloridegradient in the presence of 0.1% citrate and 0.1% PEG at pH 6.5. Thesodium chloride gradient is established between 10 and 200 mM. Thefractions containing most of the thrombin are collected, diafiltered,the concentration being thereby adjusted to approximately 500 U thrombinper mL. Such a solution can be frozen, stored after freeze-drying orfurther processed without delay.

After clarifying filtration by filters of different pore sizes rangingfrom 5000 nm to 75 nm, the thrombin-containing solution is filteredthrough a 35 nm-nanofilter and preferably through further nanofiltershaving pore sizes of 20 nm and 15 nm. The virally partitioned thrombinsolution obtained in that manner is concentrated to a content of morethan 5000 U thrombin per mL using a 30 KD-filter, frozen orfreeze-dried, and stored.

Preparation of a virally safe medicinal product capable of generatingthrombin.

An equivalent sample of the virally safe pharmaceutical substance thathas been produced according to Example 9 and has been frozen orfreeze-dried, is diluted with a 0.9% NaCl solution in a manner to ensureit will generate 500±50 U thrombin per mL after recalcification at 37°C. within two hours.

Such a diluted sample is mixed with an equal amount of a 5-10%fibrinogen-containing medicinal product which is planned to form part ofa fibrin sealant kit, and the coagulation time of this mixture isdetermined after recalcification. If the coagulation time at 37° C.exceeds 150 seconds, the pharmaceutical substance according to Example 7is admixed with an amount of thrombin produced as described in Example 8appropriate to lower the coagulation time to between 100 and 150seconds.

The amounts of thrombin determined in that manner are added to thesubstance obtained as described in Example 9 under stirring at atemperature of between 0° and 2° C., and the mixture is clarified byfiltration and sterile filtered. The resulting sterile bulk solution isportioned and filled into final containers, preferably at the sametemperature, and is freeze-dried. The freeze-dried powder isreconstituted with a sterile 5 mM calcium chloride solution.

Preparation of a virally safe medicinal product containing thrombin.

A pharmaceutical substance according to Example 10 is thawed orreconstituted and adjusted to a concentration of 0.9% NaCl using sodiumchloride. Isotonic saline is added to adjust the thrombin concentrationto between 500 and 5000 Upper mL, as desired. PEG is added in an amountto produce a 0.1% solution, and a 10% CaCl₂ solution is added in anamount to obtain a 5 mM CaCl₂ solution. This solution is sterilefiltered, portioned and filled into final containers under sterileconditions. It can be frozen or freeze-dried for storage. Freeze-driedthrombin solution is reconstituted with Water for Injections.

Use of a medicinal product capable of generating thrombin in combinationwith a medicinal product containing thrombin.

1 mL of fibrinogen-containing medicinal product containing 50-100 mgfibrinogen per mL is mixed with 1 mL of a medicinal product capable ofgenerating thrombin according to Example 11. The mixture of the twomedicinal products is stirred under sterile conditions for approximately20 seconds, and the homogenous mixture obtained in that manner isapplied to tissues to be glued or sealed after they have been adapted totheir desired positions. The glued or sealed tissues are held in theadjusted positions until the mixture of the two medicinal products hascoagulated. If necessary, they may be arrested in the fixed position fora longer period of time, even for several hours.

Use of a medicinal product containing thrombin.

To 2.0 mL of a mixture of medicinal products according to Example 13,0.2 mL of a medicinal product containing thrombin according to Example12 at a concentration of 2000 u thrombin/mL are added and mixed rapidly.To achieve hemostasis, this mixture is applied to bleeding sites withoutdelay, preferably after accumulated blood has been removed by sucking,swabbing or blowing it off.

Determination of coagulation factor XI and of coagulation factor XII inion-exchanger eluates according to Example 4a which are not stored aftercharging but eluted immediately after washing of the charged ionexchanger.

Determination is carried out using coagulation-factor-deficient plasmasby American Diagnostica.

Determination of kallikrein and prekallikrein in eluates of ionexchangers which are charged with prothrombin complexes and are elutedimmediately after washing or only after storage for up to 100 hours atrefrigerator temperature.

Kallikrein activity is determined using chromogenic substrate S-2403. A4 mM chromogenic substrate solution is used, which gives a finalconcentration of 0.4 mM. The prekallikrein concentration is alsodetermined with chromogenic substrate S-2403, a 1:100 dilution of theeluate being mixed with an equal volume of a 0.5% kaolin suspension andincubated at 37° C. under stirring for five minutes. Prior to themeasured extinction, the extinction which is obtained without activationof the kaolin suspension is deducted, and the amounts or unitsprekallikrein are determined using a standard curve.

Determination of prekallikrein activator.

Prekallikrein activator determination is performed in the same manner asthe determination of prekallikrein, except that a concentration seriesof prekallikrein activators is used instead of kaolin suspensions. Ifcopper salts of ellagic acid are used, a concentration series of between10 ng and 10000 ng is used, which generates the maximum amount ofkallikrein from a prekallikrein sample at 37° C. within five minutes.

Preparation of plant and/or or synthetic phospHlipid emulsions fromL-a-phosphatidylcholine, L-a-phosphatidyl-L-serine, andL-a-phosphatidyl-ethanolamine, which is the same as described in Example5.

Determination of TAFIa.

TAFIa is determined using chromogenic TAFI activity kits by AmericanDiagnostica without the addition of activator (thrombin). The content ofTAFIa is calculated from a standard curve, which was established usingdilutions of plasma and subsequent activation with thrombin andmeasurement of the TAFIa-activity with a chromogenic substrate.

Determination of coagulation factor XIIIa.

Determination of activated factor XIII is carried out according toEuropean Pharmacopoeia Suppl. 4.5. 07/2003, p. 3687 without the use ofactivators of factor XIII (thrombin).

Ultrasound treatment of phospholipid emulsions.

From the phospholipid mixtures, 1% suspensions are produced by dilutionwith isotonic saline, and 2 mL are subjected to ultrasonic irradiation.Ultrasonic irradiation is carried out with Ultrasound DesintegratorSonifire II W-250. The output is a maximum of 200 watts at a frequencyof 20 kHz. Konverter 102 C was used with standard resonator ½″ andmicrotip 101-148-062. Ultrasonic irradiation is carried out for between10 and 100 seconds without cooling of the irradiated emulsion at step 1and at 10% pulsating interval. The activity of the irradiatedphospholipid emulsion is determined by assessment of the partialthromboplastin time using a reference plasma and a kaolin suspension.100 μL of plasma are incubated with 50 μL of a liposome emulsion and 50μL of a 0.5% kaolin suspension at 37° C. for five minutes, recalcified,and the coagulation time at 37° C. is determined. The required amount ofliposome emulsion is determined which causes the shortest coagulationtime. The activities of the liposome emulsions described in Examples 5and 6, and of the phospholipid emulsions according to Example 18 can bedetermined by this method.

Determination of the appropriate Ca-ion concentration for the desiredadjustment of the coagulation time of a pharmaceutical substance capableof generating thrombin.

Pharmaceutical preparations generating thrombin are produced, and ageometric dilution series of the pharmaceutical preparation to be testedis mixed with Ca-ions, the highest Ca-ion concentration being 1 mM. Thedilution series ranges from 125 μM to 1000 μM. Similarly, a Ca-ionconcentration series is prepared ranging from 10 mM to 640 mM, and thecoagulation time is determined. The required Ca-ion concentration isdetermined using a standard curve.

BIBLIOGRAPHY

-   Blombäck B. Fibrinogen: Evolution of the Structure-Function Concept:    Keynote Address at Fibrinogen 2000 Congress. Annals N.Y. Acad. Sci.    2001; 936: 1-10-   Booth N. A. TAFI Meets the Sticky Ends. Thromb. Haemost. 2001; 85:    1-2-   Davie E. W., Ratnoff O. D. Waterfall sequence for intrinsic blood    clotting. Science 1964; 145: 1310-12-   Drake T. A., Morrissey J. H., Edgington T. S. Selective cellular    expression of tissue factor in human tissue. Implication for    disorders of hemostasis and thrombosis. Am. J. Path. 1989; 134:    1087-97-   Grey E. G. Fibrin as a haemostatic in cerebral surgery. Surg. Gyn.    Obst. 1915; 21: 452-454-   MacFarlane R. G. An enzyme cascade in the blood clotting mechanism    and its function as a biochemical amplifier. Nature 1964; 202: 498-9-   Mann K. G., Jenny R. J., Krishnaswamy. Cofactor proteins in the    assembly and expression of blood clotting enzyme complexes. Ann.    Rev. Biochem. 1988; 57: 915-56.-   Matras H. et. Al. Zur Klebung von Nervenanastomosen mit    Gerinnungssubstanzen. Fortschr. Kiefer-Gesichts-Chir. 1976; 112-114-   Morawitz P. Die Chemie der Blutgerinnung. Ergebn. d. Physiol. 1905;    4: 307-   Rapaport S. I., Rao L. V. M. The Tissue Factor Pathway: How it has    become a “Prima Ballerina”. Thromb. Haemost. 1995; 74: 7-17-   Siebenlist K. R., Meh D. A., Mosesson M. W. Protransglutaminase    (F-XIII) mediated cross-linking of fibrinogen and fibrin. Thromb.    Haemost. 2001; 86: 1221-8-   Spangler H. P. Gewebeklebung und lokale Blutstillung mit Fibrinogen,    Thrombin und Blutgerinnungsfaktor XIII (Experimentelle    Untersuchungen und klinische Erfahrungen). Wien. klin. Wschr. 1976;    88(4): 3-18-   Von dem Borne P. A. K., Koppelman S. J., Bouma B. N. et al. Surface    independent factor XI activation by thrombin in the presence of high    molecular weight kininogen. Thromb. Haemost. 1994; 72: 397-402-   Young F. et al. “Suture” of Wounds by Plasma-Thrombin Adhesion. War    Med. 1944; 6: 80-85

1. A pharmaceutical composition comprising: (A) human prothrombin, (B)human coagulation factors V, VIII, IX, X, which may at least partiallybe present in activated form, and human coagulation factor XIa, and (C)a mixture of phospholipids where the phospholipids are selected from thegroup consisting of choline-phospholipids, serine-phospholipids, andethanolamine-phospholipids, where the phospholipids are contained inliposomes, wherein said pharmaceutical composition (i) the presence ofan effective amount of calcium ion activity generates thrombin and (ii)when the product of (i) is mixed with a medicinal product containingfibrinogen, results in a mixture which coagulates within 30 to 300seconds and is suitable for use as a tissue glue or tissue sealant.
 2. Apharmaceutical composition according to claim 1 further comprising humancoagulation factor VII or a mixture of human coagulation factors VII andVIIa.
 3. A pharmaceutical composition according to claim 1, furthercomprising human tissue factor.
 4. A pharmaceutical compositionaccording to claim 1 wherein the prothrombin and coagulation factors V,VIII, IX, X, XIa, have been rendered virally safe by a method selectedfrom the group consisting of virus inactivation and virus partitioning.5. A pharmaceutical composition according to claim 1 which is indeep-frozen or freeze-dried form.
 6. A pharmaceutical compositionaccording to claim 1 prepared from virus inactivated,coagulation-factor-XI- and coagulation-factor-XII-containing prothrombincomplexes which are activated by kallikrein.
 7. A pharmaceuticalcomposition according to claim 1 comprising phospholipids of plantorigin or plant and synthetic origin, preferably in a ratio ofphosphatidylcholines to phosphatidylserines of 3:1 or ofphosphatidyl-cholines to phosphatidylserines tophosphatidylethanolamines of 2:2:1.
 8. A pharmaceutical compositionaccording to claim 1, wherein the comprised phospholipids are preparedusing a phospholipid emulsion which has been subjected to ultrasonicirradiation.
 9. The pharmaceutical composition according to claim 8,where the emulsion is further subjected to sterile filtration.